US6660830B1 - Peptides with antiproliferative properties - Google Patents

Peptides with antiproliferative properties Download PDF

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US6660830B1
US6660830B1 US09/155,165 US15516599A US6660830B1 US 6660830 B1 US6660830 B1 US 6660830B1 US 15516599 A US15516599 A US 15516599A US 6660830 B1 US6660830 B1 US 6660830B1
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peptide
amino acid
growth factor
peptides
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Razvan T Radulescu
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention pertains to peptides having antiproliferative properties, nucleic acids (DNA/RNA) coding for said peptides, peptide nucleic acids that are structurally homologous to said nucleic acids and the use of said peptides, said nucleic acids, said peptide nucleic acids and/or pharmaceutical compositions thereof in biotechnology, in molecular biology, bioinformatics, as well as the diagnosis and therapy of hyperproliferative diseases, particularly cancer and atherosclerosis.
  • DNA/RNA nucleic acids
  • peptide nucleic acids that are structurally homologous to said nucleic acids and the use of said peptides, said nucleic acids, said peptide nucleic acids and/or pharmaceutical compositions thereof in biotechnology, in molecular biology, bioinformatics, as well as the diagnosis and therapy of hyperproliferative diseases, particularly cancer and atherosclerosis.
  • the objective of the present invention is to provide a means to inhibit the growth of a variety of different types of cancer eventually resulting in a regression of the tumors.
  • a peptide by a nucleic acid (DNA/RNA), by a peptide nucleic acid, by a pharmaceutical composition and by the use of said peptides, by the use of the DNA/RNA, by the use of the peptide nucleic acid and by the use of a pharmaceutical composition the present invention.
  • DNA/RNA nucleic acid
  • a pharmaceutical composition by a pharmaceutical composition and by the use of said peptides, by the use of the DNA/RNA, by the use of the peptide nucleic acid and by the use of a pharmaceutical composition the present invention.
  • the present inventor has found a promising approach in the treatment of cancer which is to utilize specific peptides that block substances causing cancer, particularly oncogenic proteins.
  • the peptides of the present invention are characterized by a short sequence length which renders them economical in terms of their synthesis. Moreover, they are able to efficiently penetrate cells and, as a result, are capable of neutralizing intracellular proteins that promote the development of cancer.
  • tumor suppressor proteins such as RB1
  • RB1 Tumor suppressor proteins
  • the present inventor has found that it is sufficient to introduce parts of a tumor suppressor protein into cells in order to block growth factors that accelerate the uncontrolled growth of cells. It is not possible to introduce the entire tumor suppressor protein since such a protein is too long and is in most cases is subject to proteolytic degradation, thus preventing the protein from exerting its effect. On the other hand, if only parts of tumor suppressor proteins are utilized, said parts are too rapidly degraded as well, i.e. they are not sufficiently stable.
  • the peptides according to the present invention are therefore synthetic fusion poly-peptides consisting of the components (A) and (B), wherein these components are connected to each other as AB or BA.
  • the first component (A) comprises an effective part of a tumor suppressor protein—in such a form as first found by the inventor—or is hydropathically homologous thereto.
  • the second component (B) comprises a sequence that stabilizes the first component (A).
  • the peptide of the present invention binds to a growth factor or a growth factor receptor, preferably to the sequence LXCXE (SEQ. ID. NO: 1) therein, more preferably to the LXCXE (SEQ. ID. NO: 1) sequence in insulin, i.e. to LVCGE (SEQ.
  • IGF-1 and IGF-2 comprise the sequence FVCGD (SEQ. ID. NO: 3), which sequence is hydropathically homologous to the segment LVCGE (SEQ. ID. No: 2) in insulin (R. Radulescu & C. Wendtner, J. Mol. Recognition 1992, vol. 5, pp. 133-137) the peptide of the invention binds to IGF-1 and IGF-2 as well.
  • the component (A) is further characterized by the fact that it is hydropathically complementary to a fragment of a growth factor or of a growth factor receptor according to the complementary peptide theory (J. E. Blalock, Trends in Biotechnology, vol. 8, pp. 140-144, June 1990) or according to the three-dimensional configuration, respectively.
  • growth factors are: insulin, IGF-1, IGF-2, EGF, FGF, angiogenin, NGF and PDGF.
  • the component (AN) comprises a section of RB1, in particular amino acids 649-654 of RB1, i.e. LFYKKV (SEQ. ID. No: 4) or is hydropathically homologous thereto.
  • the component (B) of the fusion peptide has the property of stabilizing said component (A) as a cofactor so that the latter is not proteolytically degraded in the cell. This may be achieved by the peptides of the present invention rapidly arriving at the cell nucleus where they are less prone to proteolytic degradation (R. Fahraeus et al., Current Biology 1996, vol.6, no. 1, pp. 84-91) or by using branched components (B) such as polylysine branches or polylysine cores or by using D-amino acids.
  • the component (B) is a polylysine core (R. Radulescu et al., Biochemical and Biophysical Research Communications, 1995, vol. 206, pp.
  • peptides which are preferably directed to the cell nucleus
  • NLS nuclear localization sequence
  • the component (A) has the following sequence or is homologous thereto in at least two positions:
  • This peptide is a combination of the sequence P1 and the polylysine core [GGG] 4 -[KRG] 2 KG (SEQ. ID. No: 7)
  • This peptide is a combination of the sequence P1 and the polylysine core [GGG] 4 -[K] 2 -KG (SEQ. ID. No: 9).
  • This peptide is a combination of the sequence P4 and the polylysin core [GGG] 4 -[1KdRG] 2 -1KG (SEQ. ID. No: 7).
  • This peptide is a combination of the sequence P4 and the polylysine core [GGG] 4 -[1K 2 -1KG (SEQ. ID. No: 9).
  • This peptide P5 is composed of the sequence P1 (SEQ. ID. No: 4 and the nuclear localization sequence of the large T antigen of the SV40 virus.
  • This peptide P6 is a combination of the sequence P1 (SEQ. ID. No: 4) and Penetratin, a sequence consisting of 16 amino acids located within the Antennapedia homeodomain, which sequence mediates translocation across membranes and thus also nuclear localization.
  • This peptide P7s is a combination of the sequence dKdVdLdYdFdK which is hnydro-pathically homologous to P1 and the polylysine core [GGG] 4 1KdRG] 2 1KG (SEQ. ID. No: 7).
  • This peptide P7 is a combination of the sequence dKdVdLdYdFdK (SEQ. ID. No. 13), which is hydro-pathically homologous to P1 and the polylysine core [GGG] 4 -[1K] 2 -1KG (SEQ. ID. No: 9).
  • This peptide P8 is a combination of the sequence dKdVdLdYdFdK (SEQ. ID. No: 13) which is hydro-pathically homologous to P1 and Penetratin.
  • the peptides of the present invention normally comprise L- and/or D-amino acids.
  • the all-L-forms, the all-D-forms, the retro-inverse isomers and the corresponding permutations of the L- and D-form of each amino acid are within the scope.
  • all permutations of the oxidized and reduced forms of each amino acid and amino acids both in the free form and bearing protective groups are within the scope.
  • the most effective concentration of the peptides according to the present invention is in the range of from 10 ⁇ 4 to 10 ⁇ 5 M, with other effective concentrations being also possible depending on the specific application.
  • branched peptides As far as branched peptides are concerned, it has been shown to be advantageous that they are present in the all-D form. It is assumed that linear peptides containing an NLS enter the cell nucleus; branched peptides are likely to enter the cell nucleus, but they may also be effective outside the nucleus.
  • the peptides according to the present invention may be designated “SCAPs”, i,e. “Synthetic Cofactor-associated Anti-oncogenic Peptides”.
  • the preparation of the peptides according to the present invention is preferably performed according to the common solid-phase method (see G. A. Grant, “Synthetic Peptides”, W.H. Freeman and Company, New York, 1992).
  • the subsequent purification of the peptides according to the present invention is carried out and is verified as described ((R. Radulescu et al., Biochemical and Biophysical Research Comnmunications 1995, vol. 206, pp. 97-102).
  • the latter reference also provides an example for using the peptides of the present invention for the biotechnological purification of growth factors, e.g. insulin, and growth factor receptors.
  • growth factors e.g. insulin
  • growth factor receptors e.g. insulin
  • the peptides according to the present invention that carry an NLS could be coupled to a chromatography column comprising a heparan sulfate matrix.
  • the components (A) and (B) of the peptides according to the present invention may also be cleaved from the corresponding proteins and connected with each other according to standard methods. Additional techniques for the preparation of the peptides are within the ordinary skill (see G. A. Grant, “Synthetic Peptides”, W.H. Freeman and Company, New York, 1992).
  • the invention further relates to DNA/RNA coding for the peptides according to the present invention with the DNA/RNA sequence being derived from the genetic code.
  • DNA/RNA sequence codes for the above mentioned amino acid sequences (P1) and (P4) that represent component (A) of the peptide according to the present invention:
  • DNA/RNA sequences coding for the peptides of the present invention may be incorporated into adequate vectors for use in the gene therapy of cancer.
  • a survey of the methodology is to be found in R. C. Mulligan, Science 1993, vol. 260, pp. 926-932.
  • the above DNA/RNA sequences comprise DNA/RNA sequences hybridizing thereto under stringent conditions, as known to those skilled in the art. These are in particular DNA/RNA sequences that hybridize with the above DNA/RNA sequences at about 20° C. below the melting point of DNA/RNA. Moreover, the above mentioned DNA/RNA sequences comprise DNA/RNA sequences that are related to the above mentioned DNA/RNA sequences according to the degeneracy of the genetic code.
  • the present invention may also be applied in bioinformatics and molecular biology.
  • the following strategy could be applied to identify tumor suppressor proteins that interact with a growth factor or its receptor.
  • the cDNA of the corresponding growth factor or its receptor, respectively could be derived from the NCBI data base and this cDNA may be translated according to the complementary peptide strategy into a complementary DNA and into a peptide by means of the DNA Strider software.
  • homologous proteins/peptides may be found (the sought tumor suppressors) by means of the BLAST algorithm in the OWL data base navigator.
  • This method could accelerate and facilitate the cloning of (novel) tumor suppressor proteins or (novel) growth factors or growth factor receptors, respectively.
  • the cDNA of the human EGF precursor has been copied from the NCBI data base and copied into the DNA Strider software.
  • the complementary DNA sequence(s) has/have been derived from the EGF precursor cDNA and this DNA sequence(s) has/have been translated into peptides, into so-called peptides complementary to the EGF precursor protein.
  • the BLAST algorithm in the OWL data base navigator peptides/proteins were searched for that are homologous to these complementary peptides.
  • the amino acids of the EGF precursor 209-213 (REGSN) and the amino acids 305-309 of p130 (IGTLS) are hydropathically complementary to each other and are therefore potential binding sites in the assumed complex between the EGF precursor and p130.
  • the amino acid sequence IGTLS or sequences that are hydropathically homologous thereto may represent component (A) of an antiproliferative peptide of the present invention.
  • the peptides of the present invention are used in a pharmaceutical composition either alone or together with several common adjuvants, fillers and/or additives.
  • a particularly advantageous embodiment is the combination of the above mentioned peptides P3 and P6 as well as of P3 and P5, respectively.
  • the form of administration of the pharmaceutical composition is concerned, the following is appropriate: ointments, solutions, dispersions, emulsions, aerosols, foams, particulate matter (for example granulates, agglomerates, powder, micropearls, adsorbates), pills, pastilles, tablets, dragées or capsules.
  • the peptides of the present invention may also be used together with cytostatic remedies or in combination with radiation treatments.
  • the peptides are preferably administered locally, intracutaneously or transcutaneously, for systemic administration preferably intravenously, intraarterially, orally and rectally.
  • the administration into cavities is preferably carried out intrathecally, intraperitoneally or intracavitarily.
  • the peptides, DNA/RNA sequences and pharmaceutical compositions according to the present invention are useful as drugs for treating cancer and are applied according to the present invention in this manner, displaying a marked cytotoxic effect.
  • the peptide according to the present invention generally acts on all tumor cells that display a defective retinoblastoma gene or protein, respectively.
  • FIG. 1 to FIG. 12 shows the % cell population of MCF-7 cells or SAOS-2 cells in the G1-, S- and G2/M phase in the presence or absence of different peptides of the present invention
  • FIG. 13 shows the cytotoxic effect of the peptide P3 on K562 cells
  • FIG. 14 shows the cytotoxic effect of peptide P3 on CCRF-CEM cells and CCRF-CEM/ACT 400 cells;
  • FIG. 15 shows the effect of peptide P3 on normal peripheral blood mononuclear cells
  • Table 1 shows the P5- and P6-mediated inhibition of the cell cycle progression in MCF-7 cells which, by contrast, is not achieved with Penetratin;
  • Table 2 shows the P5- and P6-mediated inhibition of the cell cycle progression in SAOS-2 cells which, by contrast, is not achieved with Penetratin.
  • FIG. 1 shows that, under serum-free conditions, the peptide P3 of the present invention at a concentration of 10 ⁇ 5 M reduces the G1 phase and increases the S phase in MCF-7 cells that have been synchronized in the G0/G1 phase.
  • the morphology of the cells treated with P3 corresponds to that of apoptotic cells.
  • FIG. 2 shows that the peptide P3 of the present invention at a concentration of 10 ⁇ 5 M results in an increase of the G1 and G2/M phases as well as in a decrease of the S phase in MCF-7 cells that have been stimulated by insulin-like growth factor 1 (IGF-1) at an optimal concentration of 10 ⁇ 8 M. Accordingly, P3 blocks the effect of IGF-1 on MCF-7 cells. In particular, P3 delays the progression of the cell cycle caused by IGF-1 and thus delays cell division. The morphology of the cells treated with P3 corresponds to that of apoptotic cells.
  • IGF-1 insulin-like growth factor 1
  • FIG. 3 shows that, under serum-free conditions, each of the peptides P4, P5 and P6 at a concentration of 10 ⁇ 5 M reduces the G1 phase and increases the S phase in MCF-7 cells that have been synchronized in the G0/G1 phase.
  • the morphology of said cells, in particular of those treated with P6, corresponds to that of apoptotic cells.
  • FIG. 4 shows that the peptide P6 of the present invention at a concentration of 10 ⁇ 5 M increases the G1 phase as well as decreases the S phase in MCF-7 cells that have been stimulated with IGF-1 at an optimal concentration of 10 ⁇ 8 M. Therefore, P6 blocks the effect of IGF-1 on MCF-7 cells. In particular, P6 delays the progression of the cell cycle caused by IGF-1 and thus delays cell division. The morphology of the cells treated with P6 corresponds to that of apoptotic cells.
  • FIG. 5 shows that the combination of the peptides P3 and P5 at a concentration of 10 ⁇ 5 M each as well as the combination of P3 and P6 at a concentration of 10 ⁇ 5 M each results in an increase of the G1 phase and the G2/M phase as well as in a decrease of the S phase in MCF-7 cells that have been stimulated by 10% fetal calf serum (FCS).
  • FCS fetal calf serum
  • FIG. 6 shows that the peptide P3 of the present invention at a concentration of 10 ⁇ 5 M increases the G1- and the G2 phases as well as decreases the S phase in MCF-7 cells that have been stimulated by estradiol (E2) at an optimal concentration of 10 ⁇ 9 M or by epidermal growth factor (EGF) at an optimal concentration of 10 ⁇ 8 M.
  • E2 estradiol
  • EGF epidermal growth factor
  • FIG. 7 shows that the peptide P3 of the present invention blocks the progression of the cell cycle that is caused by IGF-1 [10 ⁇ 8 M] in a dose-dependent manner.
  • the morphology of the cells treated with P3 [5 ⁇ 10 ⁇ 6 M] and in particular of the cells treated with P3 [10 ⁇ 5 M] each corresponds to that of apoptotic cells.
  • FIG. 8 shows that, under serum-free conditions, the peptide P3 [10 ⁇ 5 M] of the present invention reduces the G1 phase and increases the S and G2/M phases in SAOS-2 cells that have been synchronized in the G0/G1 phase.
  • the morphology of the cells treated with P3 corresponds to that of apoptotic cells.
  • FIG. 9 shows that the peptide P3 [10 ⁇ 5 M] increases the G1 phase and decreases the S phase in SAOS-2 cells that have been stimulated by 10% FCS.
  • the morphology of the cells treated with P3 corresponds to that of apoptotic cells.
  • FIG. 10 shows that the peptide P3 [10 ⁇ 5 M] according to the present invention reduces the G1 phase and increases the S phase of asynchronously growing MCF-7 cells that have been incubated in DMEM cell culture medium containing 10% FCS.
  • the morphology of said cells treated with P3 corresponds to that of apoptotic cells.
  • FIG. 11 shows that the peptide P8 [10 ⁇ 5 M] of the present invention increases the G1 phase and the G2/M phase as well as decreases the S phase in MCF-7 cells that have been stimulated by IGF-1 at an optimal concentration of 10 ⁇ 8 M. Accordingly, P8 blocks the effect of IGF-1 on MCF-7 cells. In particular, P8 delays the progression of the cell cycle caused by IGF-1 and thus delays cell division. The morphology of said cells treated with P8 corresponds to that of apoptotic cells.
  • FIG. 12 shows that the peptide P7s [10 ⁇ 5 ] of the present invention increases the G1 phase and the G2/M phase as well as decreases the S phase in MCF-7 cells that have been stimulated by IGF-1 at an optimal concentration of 10 ⁇ 8 M. Accordingly, P7s blocks the effect of IGF-1 on MCF-7 cells. In particular, P7s delays the progression of the cell cycle caused by IGF-1 and thus delays cell division. The morphology of said cells treated with P7s corresponds to that of apoptotic cells.
  • FIG. 13 shows the cytotoxic effect of the peptide P3 [10 ⁇ 5 M] of the present invention on asynchronously growing K562 cells incubated in RPMI cell culture medium to be time-dependent.
  • the results have been plotted as the living cells treated with P3 being a percentage of the living cells not treated with P3.
  • the number of the living cells has been determined by counting at least 200 cells at each time point according to the trypan blue method.
  • FIG. 14 shows the peptide according to the present invention P3 [10 ⁇ 5 M] to act in a cytotoxic manner on asynchronously growing CCRF-CEM sensitive cells and CCRF-CEM/ACT 400 resistant cells that have been incubated in a RPMI culture medium containing 10% FCS.
  • FIG. 15 shows the peptide P3 of the present invention [10 ⁇ 5 M] to have no effect on normal human peripheral blood mononuclear cells, irrespective of whether they are in an quiescent (a) state or in an activated (b) state. Accordingly, the cells treated with P3 do not show any morphological changes.
  • the incubation of cells with P3 was performed for a time period of 24 hours.
  • Table 1 shows that the peptides P5 and P6 of the present invention each at a concentration of 10 ⁇ 5 M reduce the S phase in MCF-7 cells that have been stimulated by IGF-1 [10 ⁇ 8 M] or insulin [10 ⁇ 6 M], respectively.
  • the peptide Penetratin [10 ⁇ 5 M] did not show any effect, as expected.
  • Table 2 shows that the peptides P5 and P6 of the present invention at a concentration of 5 ⁇ 10 ⁇ 5 M each results in an increase of the G1 phase and a decrease of the S phase in SAOS-2 cells that have been stimulated by 10% FCS.
  • the peptides P3, P5, P6, P7s or P8 of the present invention alone and/or in combination have the property of considerably delaying the progression of the cell cycle and thus the cell division of the MCF-7 breast cancer cells, of the SAOS-2 osteosarcoma cells or of leukemia cells (K562, CCRF-CEM sensitive , CCRF-CEM/ACT 400 resistant ) under particular conditions of cell culture.
  • the data shown for IGF-1 [10 ⁇ 8 M] also apply to insulin [10 ⁇ 6 M].
  • these peptides have the potential to also be effective antineoplastic agents against the growth of cancer in vivo in human beings.
  • the experimental setting comprises experiments with the breast cancer cell line MCF-7 containing an intact retinblastoma protein and the human osteosarcoma cell line SAOS-2 containing a defective retinoblastoma protein.
  • the effect of each of the added peptides on each of the above modes of stimulation, which effect reflects the rate of cell division, is investigated.
  • the control (cells synchronized in G0/G1) is fixed at time 0 after three days of starvation.
  • the remaining cells (+/ ⁇ peptide) are fixed after 24 hours.
  • the cells are subsequently analyzed by FACS for their cell cycle distribution. Analogous methods are to be found e.g. in R. Fahraeus et al., Current Biology, 1996, vol. 6, no. 1, pp. 84-91 and L. Zhu et al., Genes & Development, 1993, vol. 7, pp. 1111-1125.
  • the second experimental setting relates to the leukemia cell lines K562 and CCRF-CEM and CCRF-CEM/ACT400.
  • 100000 asynchronously growing cells/well/ml RPMI/10% FCS have each been incubated for 48 hours with each of the peptides in 6-well plates.
  • the number of dead cells/200 counted cells is determined according to the trypan blue method (L. D. Attardi et al., The EMBO Journal, 1996, vol. 15, no. 14, pp. 3693-3701 and M. K. Reeder & H. C. Isom, Cell Growth & Differentiation 196, vol. 7, pp. 449-460) after this 48-hour incubation.
  • the more dead cells the more cytotoxic the peptide and thus the more effective the peptide.

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DE19611939 1996-03-26
DE19611939 1996-03-26
DE19653445A DE19653445C1 (de) 1996-03-26 1996-12-20 Peptide mit antineoplastischen Eigenschaften
DE19653445 1996-12-20
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WO2006101283A1 (en) * 2005-03-24 2006-09-28 Biospectrum, Inc. Development of expression vector with penetratin as a method to enhance transdermal delivery of recombinant protiens
US20060251726A1 (en) * 2005-03-14 2006-11-09 Jacki Lin Bioactive FUS1 peptides and nanoparticle-polypeptide complexes
US20150148303A1 (en) * 2013-11-26 2015-05-28 Nymox Corporation Peptides effectife in the treatment of conditions requiring the removal or destruction of cells
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NO984415L (no) 1998-09-22
AU727662B2 (en) 2000-12-21
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CA2251691A1 (en) 1997-10-02
EP0892811A2 (de) 1999-01-27

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